It is to be appreciated that any discussion of documents, devices, acts of knowledge in this specification is included to explain the context of the present invention. Further, the discussion throughout this specification comes about due to the realisation of the inventor and/or the identification of certain related art problems by the inventor. Moreover, any discussion of material such as documents, devices, acts or knowledge in this specification is included to explain the context of the invention in terms of the inventor's knowledge and experience and, accordingly, any such discussion should not be taken as an admission that any of the material forms part of the prior art base or the common general knowledge in the relevant art in Australia, or elsewhere, on or before the priority date of the disclosure and claims herein.
Attenuated Total Reflection Infrared (ATR-IR) Spectroscopy
Spectroscopy is the branch of science devoted to discovering the chemical composition of materials by examining the interaction of electromagnetic radiation with the material. Infrared (IR) spectroscopy relates primarily to the absorption of energy by molecular vibrations having wavelengths in the infrared segment of the electromagnetic spectrum, that is energy of wave number between 200 and 4000 cm−1. Raman spectroscopy relates to the inelastic scattering of monochromatic light giving wavelength shifts that depend on the molecular vibrations, having typically wave number shifts between 20 and 4000 cm−1.
The structure of almost all biological molecules includes moieties that absorb energy in the IR segment of the electromagnetic spectrum. Thus, an IR spectrum of a clinical sample is representative of its main biological components and can be in the nature of a ‘metabolic fingerprint’.
ATR is a sampling technique that can be used in conjunction with IR. ATR spectroscopy offers the advantages of being potentially portable, it is inexpensive and thus has become a very powerful tool in the analysis of biological cells and tissues. ATR also allows samples to be examined directly in the solid or liquid state without further preparation, and compared with transmission-IR, the path length into the sample is shorter, avoiding strong attenuation of the IR signal in highly absorbing media such as aqueous solutions.
In use, the sample is put in contact with the surface of a crystal having a higher refractive index than the sample. A beam of IR light is passed through the ATR crystal in such a way that it reflects at least once off the internal surface in contact with the sample. This reflection forms an evanescent wave which extends into the sample. The penetration depth into the sample depends on the wavelength of light, the angle of incidence and the indices of refraction for the ATR crystal and the medium being probed. The number of reflections may be varied. The beam is then collected by a detector as it exits the crystal.
Viral Hepatitis
Viral hepatitis is caused by one or more of the six unrelated hepatotropic viruses hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus and hepatitis G virus. Millions of deaths occur annually around the world due to hepatitis. Diagnosis is made by assessing a patient's symptoms, physical examination and medical history in conjunction with blood tests, liver biopsy and imaging. In many cases patients suffering hepatitis are not aware of symptoms, only becoming aware of the disease during routine blood tests.
But several liver diseases present with signs, symptoms or liver function test abnormalities similar to viral hepatitis. Accordingly, new diagnostic techniques that can rapidly and inexpensively distinguish between these various diseases of the liver are being sought. Preferably, new diagnostic techniques can distinguish between the various hepatitis viruses.
Human Immunodeficiency Virus (HIV)
HIV is a lentivirus that causes acquired immunodeficiency syndrome (AIDS) comprising progressive failure of the immune system. Many patients are unaware they have been infected by HIV and widespread, routine testing does not usually occur even amongst population sectors at high risk of infection.
HIV testing is initially by enzyme-linked immunosorbent assay (ELISA) carried out in duplicate to detect antibodies-positive patients. Confirmatory testing is then carried out with a more specific test (eg Western blot or immunofluorescence assay). If Western blot alone is used, a second specimen is usually collected more than a month later and retested. Nucleic acid testing such as PCR testing can also be performed can also help diagnosis.
Although these established testing regimes are very accurate, they are onerous in terms of time, labour and expense. Accordingly, new diagnostic techniques that can rapidly and inexpensively detect HIV infection are still being sought.
Malaria
Malaria is a mosquito borne disease caused by five parasitic protozoans of the genus Plasmodium, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium malariae, Plasmodium knowlesi. There are up to 1.2 million fatalities per annum and accurate and early diagnosis followed by the immediate treatment of the infection is essential to reduce mortality and prevent overuse of antimalarial drugs.
During the course of its life the malaria parasite transgresses through several developmental stages including a sexual and an asexual reproductive pathway. New technologies to diagnose malaria must be cost effective and have high sensitivity and be able to detect circulating stages of the malaria parasite namely the ring and gametocyte forms because these are the only stages present in peripheral blood circulation.
Optimally, the diagnosis of malaria in a patient is followed up by the appropriate antimalarial treatment which must be initiated immediately. Treatment should be guided by three main factors:                the identify of the infecting Plasmodium species;        the clinical status of the patient; and        the drug susceptibility of the infecting parasites as determined by the geographic area where the infection was acquired and the previous use of antimalarial medicines        
Determination of the infecting Plasmodium species for treatment purposes is important for three main reasons. Firstly, Plasmodium falciparum and Plasmodium knowlesi infections can cause rapidly progressive severe illness or death while the other species such as Plasmodium vivax, Plasmodium ovale, or Plasmodium malariae, are less likely to cause severe manifestations. Secondly, Plasmodium vivax and Plasmodium ovale infections also require treatment for the hypnozoite forms that remain dormant in the liver and can cause a relapsing infection. Finally, Plasmodium falciparum and Plasmodium vivax species have different drug resistance patterns in differing geographic regions. For Plasmodium falciparum and Plasmodium knowlesi infections, the urgent initiation of appropriate therapy is especially critical.
Efforts have also been made to investigate the potential of synchrotron Fourier Transform Infrared (FTIR) in combination with Principal Component Analysis (PCA) to differentiate between intraerythrocytic stages or the parasite life cycle based on the molecular signatures of Hz and specific lipids (Webster et al. Disciminating the Intraerythrocytic Lifecycle Stages of the Malaria Parasite Using Synchrotron FT-IR Microspectroscopy and an Artificial Neural Network, Analytical Chemistry 2009, 81, 2516-2524). Webster et al found that as the parasite matures from its early ring stage to the trophozoite and finally to the schizont stage there is an increase in absorbance and shifting of specific lipid bands.
This work demonstrated the potential of using FTIR spectroscopy as a diagnostic tool for malaria but clearly a synchrotron-based method is not suitable for field use or for routine laboratory use.
FTIR spectroscopic diagnosis relies on precise acquisition of spectra, spectral pre-processing and chemometric tools such as Artificial Neural Network analysis (Lasch et al., J. Chemometr. 20, 209-220 (2006)) or Unsupervised Hierarchical Cluster Analysis (Bambery et al., Biochim. Acta. 1758, 900-907 (2006); Wood et al., Gynecol. Oncol. 93, 59-68 (2004)).
There is enormous spectral variation between biological samples caused by a number of factors, including spectral scatter caused by preparative techniques and scattering artifacts which have hindered progress of FTIR as a clinical diagnostic tool. Furthermore, the some biological moieties have infra-red molar absorptivity characteristics that do not comply with Beer-Lambert law.
FTIR spectroscopic methods have also been used for the detection of cancerous and precancerous cells and tissues (Whelan et al., J. Biophotonics 6, No. 10, 775-784 (2013)/DOI 10.1002/jbio.201200112). In order to overcome non-Beer-Lambert infra-red absorption behaviour, simple statistical models were developed to predict the concentration of DNA in cells. However it is acknowledged in this study that the simple models developed from the study would not be adequate for complicated cells or complex mixtures of biological compounds.
In another study (Sitole et al, OMICS A J. Integrative Biol. 18(8) 513-523 (2014), mid-ATR-FTIR spectroscopic profiling of blood sera has been explored as a diagnostic for HIV/AIDS. While the system is judicative of the promise for diagnosis it is clear that problems arise with the data used due to modelling based on artifactual differences. In particular separation is observed in loadings plots due to differences in bound water and artefacts resulting due to a lack of ATR correction on the spectra.
Accordingly, there is a need for new methods to enable wider use of FTIR as a diagnostic.